The advent of recombinant DNA technology in the 1970's led to the isolation of a myriad of DNA sequences that encode useful proteins. The in vitro expression of cloned eukaryotic genes in mammalian cells is well documented. (for review, see (1) Maniatis, et al., 1990, In: Molecular Cloning: A Laboratory Manual; Vol. II, Chapter 16; (2) Bebbington and Hentschel, 1987, In: DNA Cloning Vol III: A Practical Approach, Ed., Glover, O. M., pp. 163-188; (3) Sambrook and Gething, 1988, Focus 10(3):41-48). The utilization of eukaryotic expression systems has led to an increased understanding of eukaryotic promoter strength, intron function, RNA splicing and polyadenylation functions as well as transport phenomena of newly synthesized polypeptides. However, a more practical application of in vitro eukaryotic expression systems is the cloning of novel cDNAs as well as the overproduction and isolation of novel gene products. Expression of eukaryotic proteins in eukaryotic hosts more readily allows production of functional proteins correctly folded and modified (e.g., via glycosylation and acetylation, for example).
Two basic types of mammalian expression systems have been developed to date. The first involves viral expression vectors modified to express a gene of interest (for a review, see Muzyczka, 1989, Current Topics in Microbiology and Immunology). This modification usually encompasses the replacement of a portion of the viral genome with the gene of interest. The functions lost in cis from the viral genome are complemented by cloning this deleted genomic region into a separate "helper plasmid". Therefore, co-transfection of both recombinant vectors results in propagation of the virus as well as overproduction of the protein of interest. The second type of mammalian gene expression system involves the construction of DNA plasmid vectors possessing the capacity to express cloned inserts in mammalian cells. The expression of the cloned gene may occur in a transient, extrachromosomal manner or through the stable transformation of the respective mammalian host cell line. The typical mammalian expression vector will contain (1) regulatory elements, usually in the form of viral promoter or enhancer sequences and characterized by a broad host and tissue range; (2) a "polylinker" sequence, facilitating the insertion of a DNA fragment within the plasmid vector; and (3) the sequences responsible for intron splicing and polyadenylation of mRNA transcripts. This contiguous region of the promoter-polylinker-polyadenylation site is commonly referred to as the transcription unit. The vector will also contain (4) a selectable marker gene(s) (e.g., the .beta.-lactamase gene), often conferring resistance to an antibiotic (such as ampicillin), allowing selection of initial positive transformants in E. Coli; and (5) sequences facilitating the replication of the vector in both bacterial and mammalian hosts.
An example of such an expression vector is CDM8 (Seed, 1987, Nature 329: 840-842; Seed and Aruffo, 1987, Proc. Natl. Acad. Sci. USA 84: 3365-3369; Aruffo and Seed, Proc. Natl. Acad. Sci. USA 84: 8573-8577), the parental plasmid to the pCMX plasmid of the present invention. The transcription unit of CDM8 is composed of a chimeric promoter (the human cytomegalovirus AD169 constitutive promoter fused to the T7 RNA polymerase promoter), a polylinker region and the SV40 small tumor (t) antigen splice and early region polyadenylation signals derived from pSV2. The human cytomegalovirus (HCMV) promoter is expressed in a variety of mammalian cell types, while the T7 bacteriophage DNA-dependent RNA polymerase promoter can drive in vitro cell-free transcription/translation of cloned inserts. This particular promoter fusion allows initial experiments to be conducted within the confines of the host mammalian cell type, while further analysis and utilization of the cloned insert may potentially be carried out in an in vitro "cell-free" transcription/translation system. The constitutively expressed HCMV promoter has also been utilized in other mammalian expression vectors besides CDM8 (for example, see Andersson, et al., 1989, J. Biol. Chem. 264(14):8222-8229). Origins of replication in CDM8 include (1) .pi.VX (allowing e.g., replication in E. coli) (2) SV40 origin (allowing e.g., replication in a variety of COS cell types) (3) polyoma origin (allowing e.g., replication in polyoma virus transformed mouse fibroblasts) and (4) the bacteriophage M13 origin (allowing e.g., generation of single-stranded template for DNA sequence analysis and/or oligonucleotide site-directed mutagenesis). Finally, CDM8 carries the supF gene for selection in E. coli. In this antibiotic selection system, a CDM8-based plasmid construction is transformed into a specialized E. coli strain containing an episome carrying genes encoding resistance to the antibiotics, ampicillin and tetracyline. However, both genes contain chain termination ("nonsense" codon) point mutations inactivating the resistance phenotype. The supF gene product, a nonsense suppressor tRNA, restores the resistant phenotype for each antibiotic. Therefore, selection is based on growth of the specialized episomal-carrying E. coli strain on media containing ampicillin and tetracycline. Colonies exhibiting this phenotype are supposedly transformed with the CDM8-based plasmid construction.
The CDM8 vector is compatible with COS cell lines as well as cell lines transformed with the polyoma virus. COS cell lines are African green monkey CV1 cells transformed with an origin-defective SV40 mutant virus. The COS cells produce the large T antigen, which is required in trans to promote replication of SV40 or plasmid constructions such as CDM8 which contain the respective cis-acting sequences initiating viral replication. Therefore, COS cells transfected with a CDM8-based construction will support replication of the plasmid, resulting in increased plasmid copy number and a transient overexpression of the gene of interest.
The major use of CDM8 is cDNA expression cloning and overproduction of specific proteins in a mammalian in vitro expression system. Expression cloning takes on various forms depending on the mode of detection utilized to identify the cDNA of interest (see discussion, infra). However, the initial step consists of isolating mRNA and synthesizing double-stranded deoxyribonucleic acid copies of the mRNA population (cDNAs). These cDNAs must be efficiently ligated to a plasmid or bacteriophage DNA cloning vector and transferred to the appropriate host prior to library screening and analysis. CDM8 contains two BstXI restriction sites, making it amenable to the "adaptor" linker procedure of ligating cDNAs to the vector, i.e., the use of DNA fragments blunt ended at one end (and therefore compatible for ligation with the blunt ended cDNA) but containing a non-palindromic overhang on the other end (in this instance, compatible for ligation with BstXI digested vector DNA, but not with other cDNAs).
A cDNA mammalian expression library may be utilized in several ways to pursue the identification and isolation of novel cDNAs (see Chapter 16 of Maniatis, et al., supra for a review). Briefly, cDNA libraries are transfected into the appropriate cell lines. A secreted gene product may be identified by a variety of assay techniques. Seed (1987, Nature 329:840-842) utilized the CDM8-based cDNA expression system (Seed and Aruffo, 1987, Proc. Natl. Acad. Sci. USA 84:3365-3369) to select cDNAs encoding novel surface membrane proteins. These cDNA expression proteins integrated on the cell surface and were selected by the ability of that cell type to bind to specific antibody coated dishes. Positive cell types were collected, the plasmids rescued and subsequently transformed into E. coli for further analysis.
The main drawback of CDM8 involves several problems present when utilizing the supF based antibiotic system for selecting transformed E. coli cells. First, the host E. coli strain displays a relatively high frequency of antibiotic resistance, so that there is often an unacceptably high background of bacterial colonies lacking plasmid sequences. Second, low yields of bacterial plasmid DNA derived from these host-specific strains are problematic if reversion occurs during the growth of a bacterial culture. Third, plasmid preparations frequently become contaminated with episomal DNA that contains the genes conferring resistance to ampicillin and tetracycline. Fourth, the requirement of specific genes conferring antibiotic resistance encompassed within an episome drastically reduces the E. coli host range.
A second common bacterial vector also utilized as a parental plasmid to the pCMX plasmid of the present invention is pGEM4Z (Promega Bulletin 036, 1988), which contains a pUC derived .beta.-lactamase gene and pBR322 origin of replication. pBR or pUC based plasmid constructions (Yanisch-Perron, et al., 1985, Gene 33:103-109) are widely used bacterial vectors.